Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II) can phosphorylate several smooth muscle contractile and associated regulatory proteins in vitro, affecting their functional activity. This has led to speculation regarding its role in regulating smooth muscle contractile activity in vivo, though very little is known specifically about its occurrence and activity in this tissue. The broad goals of the proposed research are to identify and characterize Ca2+/calmodulin-dependent protein kinases, other than MLCK, which are expressed in vascular smooth muscle (VSM), and to establish their cellular activation properties and functional substrates. We have identified several pools of Ca2+/calmodulin-dependent kinase activity in arterial smooth muscle. One major pool is a freely soluble form of CaM-kinase II which is similar to CaM-kinase II from brain and other sources in that it is a large multimer (550 kDa) of individual 50- 60 kDa kinase subunits. A second pool of CaM-kinase II with an as yet unknown subunit composition is associated with a myofilament-rich particulate fraction. Also identifiable in the myofilament extract is a large pool of Ca2+/calmodulin dependent kinase activity which can be resolved from the other pools of CaM-kinase II. Based on its unusually small size (34-35 kDa), activation properties, and immunoreactivity with anti-peptide antibodies to CaM-kinase II, we have hypothesized that this is a novel monomeric kinase with a structure corresponding to the catalytic/regulatory domain of a CaM-kinase II subunit or a closely related kinase. This proposal is aimed at 1.) biochemical purification and molecular identification of this novel kinase activity, and characterization of its physical and functional properties as compared to CaM-kinase II purified from VSM, 2.) identifying the expressed isozymes and subunit composition of CaM-kinase II in intact VSM using molecular and immunological approaches, 3.) assessing activation of CaM- kinase II in VSM and determining the role of autophosphorylation in the regulation of activity, and 4.) identifying protein substrates for the CaM-kinase activities expressed in VSM. These studies should help to clarify the role of CaM-kinase II and related protein kinases in VSM contractile regulation and provide general insight into the relationship between structure and function in these protein kinases.